人類陰道滴蟲之Myb2蛋白質動態性質研究

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王思寰(Szu-Huan Wang) 查詢紙本館藏

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系統生物與生物資訊研究所

論文名稱

人類陰道滴蟲之Myb2蛋白質動態性質研究
(Probing the Protein Dynamics of Myb2 from Human Trichomonas vaginalis)

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摘要(中)

陰道毛滴蟲(Trichomonas vaginalis)是一種厭氧性的寄生單細胞原蟲，具有鞭毛，主要寄生於女性的陰道、尿道、子宮，以及男性的前列腺，進而引起滴蟲性陰道炎，此疾病為世界最普遍的直接性接觸感染疾病。目前研究發現，致病的關鍵其中為黏附蛋白質所產生的細胞黏附作用。而基因ap65-1是黏附蛋白質(ap65)家族的一員，分子量為65 道爾吞。調控基因ap65-1的轉錄因子被認為是陰道毛滴蟲內的Myb1與Myb2蛋白質。
在基因ap65-1上有兩段DNA序列可供Myb蛋白質所辨認，分別為MRE1/MRE2r與MRE2f ，進而調控此基因的轉錄機制。研究中發現陰道毛滴蟲內的Myb2蛋白質可以與DNA序列MRE1/MRE2r與MRE2f結合；利用分子生物學的方法截取全長Myb2蛋白質中胺基酸序列從V40到M156的Myb2x截斷蛋白質，且保有與全長Myb2蛋白質相似的DNA結合能力。
在此，利用核磁共振的技術去探討截斷蛋白Myb2x以及分別與DNA序列MRE1/MRE2r與MRE2f結合後在分子動態上的差異性。在14.7Tesla的靜磁場下，藉由使用異核核磁共振脈衝序列量測遲緩速率(relaxation rate)和15N-1H異核交互作用增異參數(NOE)；並利用簡化光譜密度(reduced spectral density mapping)得到光譜密度函數再與結構比較蛋白質的分子動態差異性。未來，將可以繼續探討Myb2x蛋白質更多的詳細結合機制，進而在設計藥物上有所幫助。

摘要(英)

Trichomonas vaginalis, an anaerobic, parasitic flagellated protozoan resides mostly in female vagina, urethra, uterus as well as male prostate gland, is the causative agent of the most common nonviral sexually transmitted infections (STIs) in the world, human trichomoniasis. Recently, multiple surface adhesion proteins have been shown to be engaged in Cytoadherence, an essential step, of the T. vaginalis infection. The ap65-1 gene, a member of the adhesion protein 65 (ap65) multigene family, encodes for multiple homologous 65-kDa proteins, was found to be regulated by transcription factors, Myb1 and Myb2 proteins, in T. vaginalis.
Two DNA sequences, MRE1/MRE2r and MRE2f, on the gene, ap65-1, is recognized by Myb protein; this discovery deduce the possible involvement of Myb-like transcription factors related to the transcription mechanism within T. vaginalis. From various experiments, evidences show a specific interaction between full-length Myb2 protein with MRE1/MRE2r and MRE2f. We have found that a truncated fragment of Myb2, designated as Myb2x, consisting of amino acid V40–M156 displays similar DNA affinity. This fragment was employed for affinity binding and NMR structure-dynamic studies.
By NMR relaxation technology, the difference in dynamics between Myb2x free, Myb2x-MRE1/MRE2r and Myb2x- MRE2f complex forms was resolved. 15N spin relaxation rates and heternuclear (15N-1H) NOE were measured by standard pulse sequences at static magnetic field of 14.7 Tesla. The relaxation data were further analyzed with reduced spectral density mapping approach to deduce the spectral density functions: J(0), J(ωN) and J(ω0.87H) . The protein dynamics of Myb2x, as revealed by the reduced spectral density functions, are mapped onto the structures of the free and DNA-bound forms of Myb2x. The results showed that binding of DNA tighten the protein structure considerably. Such information will be useful for design of effective drugs for the treatment of human trichomoniasis.

關鍵字(中)

★ 核磁共振
★ 陰道滴蟲
★ Myb2蛋白質
★ 動態性質

關鍵字(英)

★ NMR
★ Trichomonas vaginalis
★ Myb2 protein
★ Dynamics

論文目次

摘要 Ⅰ
Abstract Ⅱ
誌謝 Ⅳ
Content Ⅴ
Figure and Table list Ⅷ
1. Introduction
1.1 Trichomonas Vaginalis 1
1.2 Adhesion Protein 2
1.3 Myb Protein 3
1.4 Specific Aim 4
2. Theory
2.1 Introduction to NMR 7
2.2 Introduction to Relaxation 9
2.2.1 Origin of relaxation 10
2.2.2 Random fields 11
2.2.2.1 Chemical shift anisotropy 12
2.2.2.2 Dipole-dipole interaction 14
2.2.3 Relaxation parameters: R1 and R2 and NOE 15
2.2.3.1 Relaxation parameters: R1 and R2 15
2.2.3.2 Relaxation parameters: NOE and Solomon's equation 16
2.3 Correlation Function and Spectral Density Function 19
2.4 Reduced Spectral Density Mapping 21
2.5 1D NMR Spectra of A Protein 23
2.6 Circular Dichroism 24
3. Experimental Design and Methods
3.1 Protein Preparation and Purification 30
3.1.1 Preparation of Myb2x protein 30
3.1.2 Preparation of Myb2x protein with N15-label 31
3.1.3 Preparation of N15-labeled Myb2x protein for Fe2+ binding 31
3.1.4 Preparation of DNA for DNA binding 32
3.2 Protein Information Detecting 32
3.2.1 SDS-PAGE 32
3.2.2 Ion exchange chromatography 32
3.2.3 Gel filtration chromatography 33
3.3 Circular Dichrosim (CD) Experiment 33
3.4 Data Acquisition and Process 34
3.5 Reduced Spectral Mapping 36
4. Results
4.1 Prediction of Myb2 Protein 38
4.2 Purification and SEC Profile of Myb2x Protein 39
4.3 Characterization of Myb2x Protein 40
4.3.1 Circular dichroism (CD) analysis of Myb2x protein 40
4.3.2 NMR analysis of free and bound forms of Myb2x protein 40
4.3.3 Interaction for Myb2-MRE1/MRE2r and Myb2-MRE2f
bound forms and Fe2+ iron by NMR analysis 42
4.4 Measure Relaxation Parameters 43
4.5 Estimation of Overall Correlation Time 45
4.6 Dynamic Parameters 45
5. Discussion 64
6. Future work 67
Acknowledgement 68
References 68

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指導教授

李弘謙、黃太煌
(Hoong-Chien Lee、Tai-Huang Huang)

審核日期

2008-7-10

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